A nutritional formulation in which non-toxic (to humans) bacteria thrive, is used to prevent and treat gastric disorders associated with Helicobacter pylori (also referred to as H. pylori) which are believed to be attacked by the non-toxic bacteria. Only particular strains of non-toxic comestible bacteria, when ingested by humans are effective against H. pylori. Optionally and preferably, the prophylactic and/or therapeutic effects of the comestible bacteria are boosted with egg yolk containing immunoglubins (antibodies) specific to H. pylori antigen (also referred to as xe2x80x9cH. pylori-antibodiesxe2x80x9d).
Much has been published regarding H. pylori which inhabits the human gastric mucosa. It is a gram-negative spiral rod-shaped bacterium having an outer membrane with four to six polar flagella which are sheathed and have bulbous ends; each H. pylori bacterium is about 0.85 xcexcm (micrometer) in diameter with an average length of 2.9 xcexcm. Known pathogenic (disease) factors of H. pylori are (i) urease (urea aminohydrolase) which is produced by the bacteria to allow it to thrive in a strong acid environment in the range from pH 1-3, (ii) flagella which provide the bacteria with mobility, and (iii) a protein-aceous outer membrane of the cells which membrane helps the cells to stick to the gastric mucosal cells. Survival of H. pylori relies upon creation of a relatively non-acidic microenvironment in the vicinity of the bacteria, and a relatively basic microenvironment is provided by the enzyme urease; the more basic the better, and the closer to neutral pH, the more difficult it is for the H. pylori to thrive. The ability to command a near-neutral microenvironment is an essential property of bacteria which effectively prevent and treat gastric disorders.
A result of interaction of H. pylori on the mucous membrane is the stimulation of numerous cytokines. The predominant immune response to infection is the production of interleukin-8 (IL-8). IL-8-induced neutrophils or macrophages are a direct cause of gastritis. To date, treatment to subdue secretion of gastric acid, for example with H2 isolator, is deemed unsatisfactory over the long term due to recrudescence which is now countered with medicines which act directly on the H. pylori. Presently, trends in the fight against infection by H. pylori may be categorized as follows: (a) development of antibiotics showing a direct effect against H. pylori, (b) development of vaccines for H. pylori, and (c) using anti-H. pylori antibodies which allow the live H. pylori to be terminated. For prophylaxis, (b) and (c) are preferred.
Bhatia et al in J. Clin. Microbiol. 27: 2328-2330, 1989, disclosed that L. acidophilus could inhibit the growth of H. pylori in vitro, and that this effect was due to lactic acid production. Midolo et al in J. Appl. Bacteriol. 79:475-479, 1995, disclosed that L. casei, L. bulgaticus, Pediococccus pentosaceus and Bifidobacterium bifidus could inhibit the growth of H. pylori in vitro, and that this effect was due to organic acids produced by these bacteria. However, as stated in European Patent Application EP 0 877 032 A1 to Kodama et al (hereafter, xe2x80x9cthe ""032 applicationxe2x80x9d), one cannot expect experiments conducted in vitro to be replicated in the stomach. An attempt to use Lactobacillus salivarius as a probiotic to inhibit growth of H. pylori is reported by Aiba et al in The Meeting of the 30th Japan Germ-free Animal Gnotobiology Society, Program and Abstracts, pp 22, Requested Title 18, xe2x80x9cNew Attempt for Inhibiting H. pylorixe2x80x9d (January 1997). They also used anti-H. pylori antibodies in the yolks of eggs of hens immunized with formalin-lidlled, whole H. pylori cells. In germ-free mice, the effectiveness of the L. salivarius was 2 to 3 orders of magnitude greater than that of the antibodies; effectiveness in the environment of the stomach of a mammal such as a normal mouse, or in the pH 1-3 of a human, was not investigated. Though the particular strain of L. salivarius was not identified, there is no reason to believe that any lactic acid bacteria will be comparably effective even in a germ-free mouse; data presented below in FIG. 1 indicate that several species of Lactobacillus show high in vitro activity, but are not as effective in vivo as others with comparably high in vitro activity. Moreover, as also shown in FIG. 1, strains which show desirably high in vitro activity, e.g. L. casei HY2743 and L. plantarum HY2207 are found to be far from equally effective in vivo.
In the prior art there are taught many immunization schedules under which growth of anti-H. pylori antibodies can be stimulated, most relevant among which are the disclosures of Japanese Patent Application Kokai No. 4-275232 to Takahashi et al, which discloses antibodies obtained in eggs of hens immunized against H. pylori whole cells as an antigen; and, the disclosure of the ""032 application which discloses antibodies obtained in eggs of hens immunized against (i) flagella of H. pylori separated from the rest of the cells; and (ii) urease of H. pylori separated from the rest of the cells, these being pathogenic factors associated with H. pylori. Antibodies obtained from either (i) or (ii), by themselves, had no noticeable effect on the number of cells in the stomach of five mice; however, (i) and (ii) in combination eliminated the H. pylori cells from the stomachs of 5 out of 5 mice. (see Table 2 in the ""032 application).
Takahashi et al teach the use of a solution of shattered or comminuted H. pylori as antigen, but the beneficial effects are relatively small because the solution additionally contains many other different proteins which appear to dilute, if not diminish or negate, the ability of the antigen to generate effective antibodies.
Furthermore, Kodama et al teach that either the anti-urease antibodies or the anti-flagella antibodies, or both together, may be used in combination with at least one organism selected from the group consisting of lactic acid bacteria, Enterococci, yeasts and Bacillus to inhibit the growth of H. pylori in the stomach, teaching that the presence of any live organism unexpectedly enhances the effectiveness of the antibodies, though the live organism, by itself, was ineffective in the environment of the stomach. In particular, Kodama states that L. acidophilus, L. casei, L. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were all reported to inhibit growth of H. pylori in vitro purportedly due to organic acids produced by these bacteria, but such effectiveness was of no help to assess the effect in the stomach. Evidence of the synergistic effect of anti-urease antibodies and Lactobacillus acidophilus administered in combination orally to H. pylori-infected mice is presented in Table 3 of EP ""032. Note however, that only one-half of the population of L. acidophilus is found after 14 days. Without considering the propriety of extrapolating those results to all live organisms tested, it is evident from results presented in Table 3 that one particular strain of L. acidophilus showed a synergistic effect with H. pylori-urease. However, one skilled in the art is unable, without undue experimentation, to reproduce the effect reported, because it is not reasonably possible to find the single strain among all the known strains of L. acidophilus which produces the synergistic result.
Confirmation of the ineffectiveness of the live organism, by itself, is stated as follows: xe2x80x9cL. acidophilus alone was almost the same as that of the control group, and there was no significant difference between the two groups, as shown in Table 3. Also, gastritis conditions were observed and L. acidophilus had no efficacy on suppressing gastritis.xe2x80x9d (see page 10, lines 49-52). The tests were performed on hairless mice (NS:Hr/ICR) having a high sensitivity to H. pylori infection. Such mice do not have the normal flora found in a BALB/c mouse which provides a better comparison with a human stomach.
Contrary to Kodama""s teaching, we found that to get the beneficial effects of a bacteria in vivo in the stomach, it is critical that we use a live bacteria which by itself is highly effective in vitro against H. pylori- and to boost its effect, to use antibodies produced by antigens of fractionated H. pylori. The term xe2x80x9cfractionated H. pylorixe2x80x9d refers to particular portions of H. pylori which portions are separated from the remainder of the cells; the separated portions are as follows: (i) urease; (ii) the outer membrane and (iii) the flagella; the remainder of the cells is discarded. Since it is not practical to conduct a very large number of in vivo experiments with H. pylori-infected mice using strains of various bacteria, we chose to use a combination of the three strains found after screening a limited number of strains set forth in Table 1 below. It is recognized that there may be S one or more specific strains of L. acidophilus, not suggested in the ""032 application, any one of which, by itself, may be effective against H. pylori not only in vivo but also in vitro; they chose to use a strain which was effective in neither.
It is now evident that, in the prior art, the problem of attacking the H. pylori in a stomach relied upon the H. pylori-antibodies collected from one or more constituents of fractionated H. pylori; or, in combination with a bacteria such as L. acidophilus used in the ""032 application, or any other bacteria which by itself had no noticeable effect in the environment of a stomach. The invention described hereunder derives from the realization that only particular strains of a genus have the ability, when ingested by humans, to survive the environment of the stomach for long enough to find the relatively less acidic zones around H. pylori attached in the stomach""s lining, and to excrete bacteriocins which would attack the H. pylori. A search was made for those bacteria which could be relied upon to provide a major portion of the desired attack, their effectiveness being supplemented with conventionally derived H. pylori-antibodies.
Further, the prior art typically obtained egg yolk powder by freeze-drying an aqueous solution, and was unconcerned with formulating a commercially marketable food fortified with egg yolk powder containing H. pylori-antibodies; this led to a lack of concern to stabilize the antibodies during spray-drying of egg yolk solution to make the powder, spray-drying to sterilize being the preferred commercial method of production of egg yolk powder. Sterilization requires spray-drying at a temperature of at least 65xc2x0 C. at which temperature the antibodies are unstable. The prior art did not provide a solution to the problem of finding a comestible, non-toxic water-soluble food ingredient which is able to stabilize the antibodies in the egg yolk solution at a pH and temperature which would not deleteriously affect the antibodies.
Despite the development of several medicines for the treatment of disorders due to H. pylori, the prior art has failed to suggest any logical basis for selecting an active strain of non-toxic, live bacteria for such treatment, except trial and error. By xe2x80x9cactive strainxe2x80x9d is meant a non-toxic strain of live bacteria which effectively kills or inhibits the growth of H. pylori grown as a lawn in a growth-conducive anaerobic or microaerophilic environment, on a medium in vitro, in an amount sufficient to provide a zone free of H. pylori, which zone is visually observable with the naked eye. Particularly because it was found that certain strains of lactobacillus were effective for our purpose, as set forth in Ser. No. 09/498,668, now U.S. Pat. No. 6,329,002 we chose to study additional strains to find specific strains which produced the appropriate H. pylori-specific bacteriocins, and specifically those strains which could be delivered in food routinely consumed by humans.
A natural or synthetic food is supplemented with particular strains of live bacteria which by themselves are effective against Helicobacter pyloti (also referred to as H. pylori) not only in vitro but in vivo. The effectiveness of such bacteria is strain-specific, that is, a specific strain of a species may be effective while other strains of that same species are not. Effectiveness of xe2x80x9cactive strainsxe2x80x9d identified herein may be preserved and/or enhanced with egg yolk containing antibodies specific to H. pylori antigens, so that consuming the food will prevent and/or treat gastritis, and/or gastric and duodenal ulcers.
It has been discovered that specific strains of non-toxic (to humans) live bacteria, by themselves, when maintained as xe2x80x9cactive strainsxe2x80x9d in comestible foods, such as yogurt and other bacteria-tolerant foods which may contain other living organisms, have the unique ability to imbue such foods with prophylactic and/or therapeutic properties because, it is hypothesized, active strains produce bacteriocins which directly attack H. pylori; they also compete for adhesion sites and produce organic acids which create a hostile environment for the H. pylori. In the genus of lactic acid bacteria, only the strains Lactobacillus acidophilus 1-2 HY2177, and Lactobacillus casei HY2743 (together referred to herein as xe2x80x9cactive lactic acid bacteria strainsxe2x80x9d or xe2x80x9cactive strainsxe2x80x9d for brevity) have been found either to minimize the growth of, or to destroy H. pylori not only in vitro but also in vivo in the environment of a stomach, provided each strain is used, either individually or in combination, in an effective dosage amount. Their effectiveness is measured by the ability of these active lactic acid bacteria strains (i) to inhibit the attachment of H. pylori to gastric mucosal cells of the stomach""s mucous membrane; (ii) to inhibit the production of urease substantially completely, indicated by no significant increase in pH, and (iii) to inhibit the production of Interleukin-8 (IL-8) by as much as about 90%, indicated by an ELISA analysis.
It is therefore a general object of this invention to provide a food for general human consumption, comprising a food stored at a temperature in the range from about xe2x88x9245xc2x0 C. but no more than 45xc2x0 C. and effective to inhibit and/or prevent the growth of H. pylori in a human stomach; the food is fortified with an active strain selected from the group consisting of Lactobacillus acidophilus HY2177 and Lactobacillus casei HY2743, preferably a combination of both, optionally in combination with antibodies obtained in the yolk of an egg of a hen immunized against a pathogenic factor selected from the group consisting of fractionated H. pylori and urease of H. pylori. xe2x80x9cFractionated H. pylorixe2x80x9d consists essentially of a pathogenic factor selected from the group consisting of H. pylori-urease (hereafter referred to only as xe2x80x9cureasexe2x80x9d), flagella of H. pylori and outer membrane of H. pylori separated from the rest of the mass of H. pylori cells. Portions (ii) and (ii) are typically used together (referred to as xe2x80x9cflagella/outer membranexe2x80x9d) to avoid the effort of separating them, were each to be used individually. Urease, though not an integral portion of the H. pylori cells is referred to as a constituent of fractionated H. pylori because it is derived from, and separated from the comminuted cells.
It is a specific object of this invention to provide an improved food selected from the group consisting of a conventional food of lactic acid bacterial origin and a non-conventional food, in combination with an effective dosage amount of an active strain selected from the group consisting of Lactobacillus acidophilus HY2177 and Lactobacillus casei HY2743, the conventional food being selected from the group consisting of yogurt, buttermilk cream cheese and ice cream, and the non-conventional food being a nutritional yogurt drink. The useful concentration of active lactic acid bacteria strain(s) in the food is in the range from about 1xc3x97108 cfu/ml to about 1xc3x971010 cfu/ml, preferably in the range from 5xc3x97108 cfu/ml to about 5xc3x971010 cfu/ml, and optimally about 1xc3x97109 cfu/ml in a unit serving. The useful concentration of H. pylori-antibodies in egg yolk is in the range from 50 mg to about 250 mg per unit serving, typically in yogurt. The egg yolk is typically used in a fortified food in the range from about 0.05% to about 4% by weight of the food, preferably in the range from 0.5 to 2%, so that at 2% the food contains less than 125 mg of antibodies.
It is another specific object of this invention to provide a food or food supplement containing an effective dosage amount of at least one of L. acidophilus HY2177 and L. casei HY2743, which over a course of treatment, effectively (i) inhibits the attachment of H. pylori to gastric mucosal cells of the stomach""s mucous membrane; (ii) inhibits the production of urease substantially completely, indicated by no significant increase in pH, and (iii) inhibits the production of Interleukin-8 (IL-8) by as much as about 90%, indicated by an ELISA analysis.
It is another general object of this invention to provide a method of preventing and/or treating disorders associated with infection by H. pylori, the method comprising administering to a human a nutritional food in combination with an effective dosage amount of one or more of the active strains identified immediately hereinabove, in plural successive unit servings, each unit serving spaced apart from a prior one by a period in the range from 1 hour to 3 days; and, preferably to do so in combination with egg yolk powder containing H. pylori-antibodies derived from one or more of the aforespecified pathogenic factors, most preferably both in combination, wherein the antibodies have been stabilized with from about 5% to about 20% by weight of a water-soluble simple sugar. By a xe2x80x9cunit servingxe2x80x9d we refer to a conventional individual serving for the particular food being fortified; for example, for yogurt, a typical unit serving is 8 fl oz (fluid ounces) or about 450 ml, though it may range from 4 fl oz to 12 fl oz (250 ml to 950 ml). The term xe2x80x9csimple sugarxe2x80x9d refers to a carbohydrate containing an xcex1-hydroxy aldehyde or an xcex1-keto primary alcohol, preferably sucrose, fructose, lactose, glucose, dextrose and the like.
It is another specific object of this invention to provide a novel adjuvant which is more effective to potentiate an immune response to H. pylori antigens than conventionally used Freund""s adjuvant (complete or incomplete) and which not only produces more antibodies than with conventional adjuvants, but also produces more than 75% of the antibodies in the yolk of a hen immunized with an antigen of fractionated H. pylori in the novel adjuvant.